Synthesis, Characterization and Cytotoxicity Studies of Aminated Microcrystalline Cellulose Derivatives against Melanoma and Breast Cancer Cell Lines

Abstract

Cellulose based materials are emerging in the commercial fields and high-end applications, especially in biomedicines. Aminated cellulose derivatives have been extensively used for various applications but limited data are available regarding its cytotoxicity studies for biomedical application. The aim of this study is to synthesize different 6-deoxy-amino-cellulose derivatives from Microcrystalline cellulose (MCC) via tosylation and explore their cytotoxic potential against normal fibroblasts, melanoma and breast cancer. 6-deoxy-6-hydrazide Cellulose (Cell Hyd) 6-deoxy-6-diethylamide Cellulose (Cell DEA) and 6-deoxy-6-diethyltriamine Cellulose (Cell DETA) were prepared and characterized by various technologies like Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR), nuclear magnetic resonance spectroscopy (NMR), X-ray diffractogram (XRD), Scanning Electron microscopy (SEM), Elemental Analysis and Zeta potential measurements. Cytotoxicity was evaluated against normal fibroblasts (NIH3T3), mouse skin melanoma (B16F10), human epithelial adenocarcinoma (MDA-MB-231) and human breast adenocarcinoma (MCF-7) cell lines. IC₅₀ values obtained from cytotoxicity assay and live/dead assay images analysis showed MCC was non cytotoxic while Cell Hyd, Cell DEA and Cell DETA exhibited noncytotoxic activity up to 200 μg/mL to normal fibroblast cells NIH3T3, suggesting its safe use in medical fields. The mouse skin melanoma (B16F10) are the most sensitive cells to the cytotoxic effects of Cell Hyd, Cell DEA and Cell DETA, followed by human breast adenocarcinoma (MCF-7). Based on our study, it is suggested that aminated cellulose derivatives could be promising candidates for tissue engineering applications and in cancer inhibiting studies in future.

Keywords: MCC; aminated cellulose; breast cancer cell lines; cellulose; cytotoxicity; melanoma; tosylated cellulose.

Figure 1

Characterization of cellulose and cellulose amine derivatives: (a) FTIR; (b) XRD; (c) ¹HNMR: (i) MCC, (ii) Tosylated Cellulose (CTOS), (iii) Cell Hyd, (iv) Cell DEA, (v) Cell DETA; (d) Zeta potential measurement.

Figure 2

Scheme of Synthesis of Microcrystalline Cellulose (MCC) Derivatives: (a) Tosylated cellulose (CTos) synthesis, (b) synthesis of 6-deoxy-6-hydrazide Cellulose (Cell Hyd), (c) synthesis of 6-deoxy-6-diethylamide Cellulose (Cell DEA), (d) synthesis of 6-deoxy-6-diethyltriamine Cellulose (Cell DETA).

Figure 3

Characterization of Cellulose Derivatives SEM and EDX (Analysis) in solid state of derivatives confirming the synthesis of the derivatives. (a) SEM images were taken at 20 µm, 5 µm, 2 µm and 500 µm. (b) EXD Analysis (i) MCC, (ii) C_TOS, (iii) Cell Hyd, (iv) Cell DEA, (v) Cell DETA.

Figure 3

Characterization of Cellulose Derivatives SEM and EDX (Analysis) in solid state of derivatives confirming the synthesis of the derivatives. (a) SEM images were taken at 20 µm, 5 µm, 2 µm and 500 µm. (b) EXD Analysis (i) MCC, (ii) C_TOS, (iii) Cell Hyd, (iv) Cell DEA, (v) Cell DETA.

Figure 4

Cell Culture Evaluating the IC₅₀ values for the Cellulose and its derivative MCC, Cell Hyd, Cell DEA and Cell DETA ± average standard deviation (* p < 0.05, ** p < 0.01, and *** p < 0.001) against Normal. (a) NIH3T3 and Cancer cell lines. (b) B16F10. (c) MDA-MB-231. (d) MCF-7. (e) IC50 values graph. (f) Swelling ratios of cell derivatives.

Figure 4

Cell Culture Evaluating the IC₅₀ values for the Cellulose and its derivative MCC, Cell Hyd, Cell DEA and Cell DETA ± average standard deviation (* p < 0.05, ** p < 0.01, and *** p < 0.001) against Normal. (a) NIH3T3 and Cancer cell lines. (b) B16F10. (c) MDA-MB-231. (d) MCF-7. (e) IC50 values graph. (f) Swelling ratios of cell derivatives.

Figure 5

Cell culture live dead assay after treatment with cellulose and its derivatives MCC, Cell Hyd, Cell DEA and Cell DETA, at 3 concentrations after 24 h; against Normal (a) NIH3T3 and Cancer cell lines (b) B16F10, (c) MDA-MB-231, (d) MCF-7. Live cells show green fluorescence when stained with calcein-AM, while dead cells show red fluorescence stained with ethidium homodifier. Live cell percentage is shown on the images as total percentage of live cells from four random fields with mean ± standard deviation of 3 replicates of samples. Scale bar is 100 µm.